Anti-ageing effects of a new Dimethylaminoethanol-based formulation on DGalactose induced skin ageing model of rat

YAN Bing-jian，YUAN Feng，ZHAO Cai-ling，LIU Su

（1.Department of burn and Plastic Surgery，The Affiliated Hospital of Medical School，Qingdao University，School of Medicine，Qingdao University，Qingdao 266003，Shandong，China；

2.Technical Director，Beijing Biodegradable Materials Engineering Laboratory，Beijing 100022，China；3.Department of Burn and Plastic Surgery，Laiwu People's Hospital，Laiwu 271100，Shandong，China；4.Department of Plastic and Aesthetic Surgery，The Affiliated Hospital of Medical School，Qingdao University，Qingdao 266003，Shandong，China.）

·International Communication·

Anti-ageing effects of a new Dimethylaminoethanol-based formulation on DGalactose induced skin ageing model of rat

YAN Bing-jian1，YUAN Feng2，ZHAO Cai-ling3，LIU Su4*

（1.Department of burn and Plastic Surgery，The Affiliated Hospital of Medical School，Qingdao University，School of Medicine，Qingdao University，Qingdao 266003，Shandong，China；

2.Technical Director，Beijing Biodegradable Materials Engineering Laboratory，Beijing 100022，China；3.Department of Burn and Plastic Surgery，Laiwu People's Hospital，Laiwu 271100，Shandong，China；4.Department of Plastic and Aesthetic Surgery，The Affiliated Hospital of Medical School，Qingdao University，Qingdao 266003，Shandong，China.）

Background Dimethylaminoethanol has been widely used to fight against wrinkles，in the field of aesthetic medicine there is an increasing demand for safe and effective Dimethylaminoethanol-based products to counteract the ageing process.Objective To evaluate the anti-ageing effects of a new DMAE-based formulation.Methods30 male rats were randomly allocated into treatment，D-gal ageing modeland control groups，each of which contained ten rats. Treatment group and D-gal ageing model group were subcutaneously injected with D-galactose prepared in normal saline 125mg·kg-1·d-1for 42d.Control groups were injected with normal saline for 42 d with same method and dose.From the 18th day，after shaving their hair，the treatment grouprats were injected thisnew DMAE-based formulation at a dose of 1ml per week for 4 weeks in the Dermis of two sides hip skin mark zone.Meanwhile，D-gal ageing model group rats were administrated the same volume of normal saline with same method.Skin specimens were obtained 3days after the last treatment.Dermal collagen density and dermal thickness were evaluated by H&E and Massontrichrome staining.And mRNA expressions of TGFβ1，Smad3，Type I，Type III Pro-collagen，TIMP-1，MMP-1，were assessed by Real-time quantitative polymerase chain reaction.Results Dermal thickness，dermal collagen density and hydroxyproline content in treatment group increased significantly comparing with D-gal ageing model group.No differences were found in mRNA expression of MMP-1 and Type III Pro-collagen between the treatment group and D-gal ageing model group.In addition，mRNA expression of TGFβ1，Type I Pre-collagen，TIMP1 and smad3 in treatment group were significantly up-regulated in contrast with D-gal ageing model and control group.Conclusion This new DMAE-based formulationcould generate anti-ageing effects by activating collagen synthesisthrough TGF-β1/Smads signaling pathway.

anti-ageing；TGF-β1/Smads signaling pathway；Dimethylaminoethanol

1 Introduction

Ageing is a complicated natural phenomenon and an inevitable spontaneous process.Skin ageing is an important sign of bodily ageing and it is influenced byintrinsic and extrinsicfactors，such as genetics，environmental exposure，hormonal changes and metabolic processes.The emergence of skin ageing is mainly manifested on altrerations of the dermal connective tissue［1］.Collagens are the major fibrillar molecules in dermal extracellular connective tissue matrix（ECM），and type Icollagen（COL I）is the most abundant structural protein in adult human dermis.The lack and atrophy of collagen could induce the appearance of skin ageing.Wrinkle，irregular pigmentation，dryness，red blotchesand decreased elasticity are all features of skin ageing，which caused by the variations in COL I composition［2］.Thus，one important strategy of anti-skin-ageing is to accelerate collagen production and suppress its degradation.So Regulation of dermal f1broblasts collagen gene expression is a significant biological event and closely associated with the pathophysiology of skin connective tissue diseases and ageing［3-5］. TGF-β1 has been long understood as the most potent direct stimulator in collagen synthesis，organ fibrosis and induces scarring through activating its downstream Smads signaling pathway［6-7］.TypeⅠ/Ⅲ precollege and tissue inhibitor of MMP-1（TIMP-1），are the downstream targets of TGF-β/Smads signaling in Dermal Fibroblasts［8］.

Mesotherapy as a kind of minimally invasive injection technology has been widespread usded in Europe and the United States，which brought up and utilized by Dr.michelPistorin in1976.Over the past decade mesotherapy has become as an intriguing method for the body contouring.This new and simple method aims to approximate the location of the therapeutic to the place of the pathology for greater efficiency and lesser side efficiency.

Various medicines and cosmeceuticals are used to anti-ageing.But the results have been shown to be unsatisfied thus far.Now，DMAE as a new anti-ageing component has been widely used in the fight against skin-ageing.It has been shown that 3% DMAE facial gel is efficacious in mitigating forehead lines and periorbital fine wrinkles by promoting the typeⅠcollagen synthesis.However，it was reported that high dose DMAE could induce vacuolization in human fibroblast cellscultured in vitro［9］.There is an increasing demand for safe，minimally invasive and effective anti-ageing DMAE-based products to counteract the ageing process in the field of aesthetic medicine.Some recent research revealed that mixtures of lowdose DMAE（0.1%）and amino acid compounds could generate marked safe anti-ageing effects on D-galactose induced skin ageing.But mere low-dose DMAE（0.1%）or amino acid compounds couldn't generate obvious antiageing effect［10］.So we speculate that low-dose of DMAE initiates the collagen synthesis process，and amino acid compounds provide raw material for collagen synthesis.

In order to evaluate potential anti-ageing effects of a new DMAE-based formulation（Cellulift○R，a new patented anti-ageing pharmaceutical product composed of DMAE（0.1%），vitamins，amino acids and minerals，etc.），our research was carried out.In the researchthe new formulation was administered intradermally by mesotherapy，the changes of histological structure and the mRNA expressions of Type I，Type III Pro-collagen were investigated. Furthermore，the molecular mechanism of the anti-skin-ageing effect provided by the DMAE-based formulation was also explored.The results would be presented herein.

2 Materials and Methods

2.1 Animals

30 male adultWistarratsweighting（220±20）g were purchased from Institute of pharmaceutical Sciences（Qingdao，China）.All experiments were performed in accordance with the guidelines on Ethical Standards for investigations in animals.The rats were housed in polycarbonate cages containing hardwood chip bedding at an air-conditioned room with the temperature maintained at 25-27℃，relative humidity at 60±5%and a 12-h light/darkcycle.The animals were acclimatized for 7 d before the experiment.All rats were randomly allocated into treatment，D-gal ageing model，and control groups，each of which contained ten rats.Treatmentand D-gal ageing model groups were subcutaneously treated by mesotherapy with D-galactose（125mg·kg-1·d-1）for 42 day［10］. Contrlgroupwas injected with normal saline for 42 d with same method and dose.From the 18th day，after shaving their hair，the treatment groupwas injected with the DMAE-based formulation at a dose of 1ml per week for 4 weeks in the dermis of both sides hip skinmark zone.Meanwhile，the D-gal modle group was administrated with the same volume of normal saline with same method。

2.2 Tissue preparation and microscopic measurement

Skin specimens were obtained 3days after the last treatment.Rats were anaesthetized by intraperitoneal injection with 10%Chloral hydrate prepared in normal saline at a dose of 0.25ml/100g.Part of the specimen was fixed in 10%buffered formalin for histological analyses.while，the other skin speciments was collected in sterile tube immediately and stored in liquid nitrogen.Rats were euthanized after the collection of the skin specimens.

Histological analyses:After staining with haematoxylin and eosin（H&E），we taken the pictures from each section under 200*magnif1cation.Dermal thickness（distance between the subcutaneous fat and the dermoepidermal junction）was measured randomly at five sites on each picture with the IPP（image pro plus）software.After Massontrichrome staining，5 different views were also randomly chosen under 400×magnification，and dermal collagen density of each view were measured with the IPP（image pro plus）software.

2.3 Real-time quantitative polymerase chain reaction

The mRNA expressions of TGF β 1，Smad3，Type I，Type III Pro-collagen，tissue inhibitor of matrix metalloproteinase-1（TIMP-1），matrix metalloproteinase-1（MMP-1）were analysed by Real-time quantitative polymerase chain reaction.Total RNA was isolated from skin samples by using TRIzol reagent（Takara Biotechnology/Takara Bio，Dalian，China）and the concentration and purity of the total RNA were determined by measuring the OD260and OD280 ratio.1.25 μg of RNA was converted to cDNA by the reverse transcriptase reagen（tRR-370 Takara Biotechnology/Takara Bio，Dalian，China）.Quantitative polymerase chain reaction（PCR）analysis was performed in Roch light Cycler 96 according to the manufacturer's Instructions with the following conditions: denaturation at 95℃for 30 s，primer annealing at 60℃for 35S，and extension at 65℃for 30 s. Primers for PCR（Table 1）were designed and synthesized by Sangon Biotech Co.，Ltd（Shanghai，China）.Analysis of Relative Geneexpression Data Using the 2-△△CTMethod［12］.

2.4 Hydroxyproline Content

Hydroxyproline content in skin was measured according to the manufacture protocol（Nanjing Jianchen Biological Engineering Co，LTD，Nanjing，China）.

2.5 Statistical Analysis

All the result was analysised by using the SPSS software（SPSS，IBM，version17.0）and Statistical analysis was performed by one-way analysis of variance（ANOVA）.Numerical data were expressed as means±standard deviation（SD）and P values＜0.05 were considered statistically significant.

3 Results

3.1 Effects of the DMAE-based formulation on the Dermal thickness and dermal collagen density

To determine the effects of the DMAE-based formulationon intrinsically aged skin，haematoxylin and eosin（H&E）and Massontrichrome staining were performed，and the dermal thickness and dermal collagen density were measured in this study（result shown in Figure 1，2）.In D-gal modle and treatment group）both dermal thickness and dermal collagen density were significantly lower than control group.But the dermal thickness and dermal collagen density of treatment group were markedly higher compared with D-gal modle group（P＜0.05）.

3.2 The effect of the DMAE-based formulation on Hydroxyprolinecontent

We found that the skin hydroxyproline content in D-gal ageing model group decreased significantly in contrast with control group.The skin Hydroxyproline content of treatment group increased significantly compared with D-gal ageing model group（P＜0.05），but it was still lower than the equivalent level of control group（P＜0.05（Figure 3）.

3.3 Transforming growth factor-β（TGFβ1），Smad3，and Type I，Type III Procollagen，TIMP-1，MMP-1mRNA expression in fibroblast

To investigate whether or not this new DMAE-based formulation modulates TGF-β1，Smad3，and Type I，Type III Procollagen，TIMP-1，MMP-1 mRNA expression，Real-time quantitative polymerase chain reaction was performed.2-△△CTmethod was used to statistical analysis the result.As shown in table 2，the expressions of TGF-β1，Smad3，and Type I Procollagen，TIMP-1 mRNA in D-gal ageingmodel group significantly decreased compared with control group.But，injection of low-dose D-galactose 42 days significantly up-regulated expression of MMP-1 mRNA.Compares the D-gal ageing model group with treatment group，we found that administration of formulation injection significantly up-regulated the mRNA expression of TGF-β1，Type I Precollagen，TIMP1 and smad3（P＜0.05），but，there was no significantly difference of MMP-1mRNA expression between two groups.In addition，there was no significant difference of Type III Precollagen expression among all groups（Figure 4）.

4 Discussion

With the improvement of living standards all over the world，people are increasingly concerned about their appearance.Postponing skin ageing becomes a hot topic.Various medical treatments and cosmeceuticals are used to anti-ageing.But the results have beenunsatisfactory thus far.There is an increasing demand for safe，minimally invasive and effective anti-ageing products to counteract the ageing process in the field of aesthetic medicine.

D-galactose is a reducing sugar that can form advanced glycation end products（AGE）in vivo，It has been shown that an elevated level of AGE in vivo may accelerate the ageing process［13］. Long-term injection of low dose D-galactose into rats could induce the high intracellular galactose level.The ageing rats shows neurological impairment，decreased activity of anti-oxidant enzymes，and poor immune responses［14］.These changes are similar to the process of nature ageing So the subacuteageing model caused by D-galactose is widely used in ageing research and drug testing.In our study，Rats subcutaneously injected with D-galactose 42 days showed obvious symptoms of ageing such as body weight change listlessness，lag in response，slow movement，and withered and lackluster fur，sagged skin.Furthermore，D-gal ageing model group rats skin indicated lower levels of hydroxyproline content and mRNA messages for type I pro-collagen，and TIMP-1，as well as the higher levels of the MMP-1 mRNA expression compared to normal skin tissue.The data above mentioned suggests that injection of 125mg/kg/d D-galactose for 6 weeks is technically feasible to create a subacuteageing model for mimicking human ageing skin.In short，ageing models were made successfully in our study.

The emergence of the ageing of the skin is mainly manifested on the change of the dermal connective tissue［1］.Dermal collagen changes related to ageing are mainly manifested on the decreased dermal thickness and spatial density of collagen.Collagen fibrils are responsible for mechanical strength and resiliency of skin［15-16］. Procollagen is the precursor of collagen；therefore，an increased production of procollagen results in an increased deposition of collagen fibres.Hydroxyproline is a fundamental characteristic component of collagen，and its content as a main and sensitive index can evaluate the skin ageing condition directly［17］.

It is known that collagen degradation and synthesis are regulated by many inflammatory factors and cytokines in skin tissues.TGF-β1 as a key fibrogenic mediator plays an improtant role in cell proliferation，growth，differentiation，tissue remodeling and wound healing，and it also plays a critical role in organ fibrosis，ECM production，accumulation of extracellular matrix and inhibition of collagenase activity［18］.It is now established that the binding of TGF-β1 to its receptor II（TβRII）can activate the TGF-β receptortypeI（TβRI）-kinase，resultingin phosphorylation Smad3［6］.Previous studies welldemonstrated that Smad3-deficiencecould suppress collagen deposition，and the expression of profibrotic TGFβ1 target genes［19］. So smad3 is the critical mediator of TGF-β1/ Smads signaling in fibrosis.Furthermore，It is now well accepted that Smad3 is acritical downstream mediator responsible for the biological effects of many fibrogenic genes，suchas ColIa1，ColIa2，ColVa2，ColVIa1，and ColVIa3［8］.Previous research also showed that increased expression of TGF-β1 and smad3 is often associated with fibrotic and ECM accumulation states［20］.So the Collagen synthesis regulating is correlated with TGF-β1/smad3 pathway.In our study，4 weeks administration of the DMAE-based formulationsignificantly upregulated the mRNA expressions of TGFβ1 and smad3.which suggested that the DMAE-based formulation administration may play a role in the up-regulation of TGF-β1 and smad3 at the transcriptionallevel.Therefore，theabovedata indicate that DMAE-based formulation can obviously promote the typeⅠcollagen synthesis through the up-regulation effect on TGFbeta1/ Smad3 signaling pathway.

We further examined the mRNA expressions of MMP-1 and TIMP-1.MMP-1 initiates collagen degradation by cleaving a single site near the C terminal end of type I collagen fibrils［21-22］.TIMP-1 was recognized as an inhibitor of various MMP-1 activities，and the imbalances of MMPs/TIMPs cause excessive ECM degradation［23］.Recent studies also find that the TIMP-1 gene is immediate-early targets of the TGF-β1/Smad3 signaling cascade［8］.More interestingly，it was observed in RT-PCR results that this new DMAE-based formulation couldn't decrease the MMP-1 mRNA expression in ageing skin，but greatly elevated the TIMP-1gene expression.So Our data indicated that the antiageing effect of this new DMAE-based formulation on TGF-β1 signaling to the protection of skin from ageing would be acceptable from the viewpoints of the increasing of TIMP-1 expression and of the suppression of MMP-1 activity without a changing in MMP-1 expression.

5 Conclusion

In summary，this study provides the first evidence that this new DMAE-based formulation may be promising for skin rejuvenation by increasing type I collagen synthesis and improving dermal structure.And the underlying mechanism of this anti-ageing effect might be regulating the TGF-β1/Smad signal pathway.In addition，we also found that the DMAE-based formulation could influence TGF-β-mediating MMP-1/TIMP-1 balance in collagen metabolism and eventulally result in inhibition of the ECM degradation.

6 Conflict of Interests

The authors declare that there is no conflict of interests with any financial organization regarding the material discussed of this paper. We thank EME for the donation of CelluliftR.

［1］Yankelevich D.Estrogen and skin［J］.Climacteric，2011，14（5）: 601.

船型组合②：A、B泊位停靠3000DWT杂货船：15 + 108 + 50 +125 + 22.5 = 320.5m ＞ 300m

［2］Calleja-Agius J，Muscat-Baron Y，Brincat MP.Skin ageing［J］. Menopause Int，2007，13（2）:60-64.

［3］Uitto J，Perejda AJ，Abergel RP，et al.Altered steady-state ratio of type I/III procollagen mRNAs correlates with selectively increased type I procollagen biosynthesis in cultured keloid fibroblasts［J］.Proc Natl Acad Sci U S A，1985，82（17）:5935-5939.

［4］Gordon MK，Hahn RA.Collagens［J］.Cell Tissue Res，2010，339（1）:247-257.

［5］Song KC，Chang TS，Lee H，et al.Processed Panax ginseng，Sun Ginseng Increases Type I Collagen by Regulating MMP-1 and TIMP-1 Expression in Human Dermal Fibroblasts［J］.J Ginseng Res，2012，36（1）:61-67.

［6］Lan HY.Diverse roles of TGF-beta/Smads in renal fibrosisand inflammation［J］.Int J Biol Sci，2011，7（7）:1056-1067.

［7］Ren ZP，Sun LP，Xia YC，et al.Effect of the protease inhibitor MG132 on the transforming growth factor-beta/ Smad signaling pathway in HSC-T6 cells［J］.J Huazhong Univ Sci Technolog Med Sci，2013.33（4）:501-504.

［8］Verrecchia F，Chu ML，Mauviel A.Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach［J］.J Biol Chem，2001，276（20）:17058-17062.

［9］Gragnani A，Giannoccaro FB，Sobral CS，et al. Dimethylaminoethanol affects the viability of human cultured fibroblasts［J］.Aesthetic Plast Surg，2007.31（6）:711-718.

［10］Liu S，Chen Z，Cai X，et al.Effects of dimethylaminoethanol and compound amino acid on D-galactose induced skin ageing model of rat［J］.Sci World J，Doi:10.1155/2014/507351.

［11］Mateoiu C，Pirici A，Bogdan F.Immunohistochemical nuclear staining for p53，PCNA，Ki-67 and bcl-2 in different histologic variants of basal cell carcinoma［J］.RomJ Morphol Embryol，2011，52（1 Suppl）:315-319.

［12］Schmittgen TD，LivakKJ.Analyzingreal-time PCR data by the comparativeC（T）method［J］.NatProtoc，2008，3（6）:1101-1108.

［13］Song X，Bao M，Li D.Advanced glycation in D-galactose induced mouse ageing model［J］.Mech Ageing Dev，1999，108（3）:239-251.

［14］Hao L，Huang H，Gao J，et al.The influence of gender，age and treatment time on brain oxidative stress and memory impairment induced by D-galactose in mice［J］.Neurosci Lett，2014，571:45-49.

［15］Zouboulis CC，Adjaye J，AkamatsuH，et al.Human skin stem cells and the ageing process［J］.Exp Gerontol，2008，43（11）:986-997.

［16］Smith JG Jr，Davidson EA，Sams WM Jr，et al.Alterations in human dermal connective tissue with age and chronic sun damage［J］.J Invest Dermatol，1962，39:347-350.

［17］FisherGJ，ChoiHC，Bata-CsorgoZ，etal.Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo［J］.J Invest Dermatol，2001，117（2）:219-226.

［18］Kirmaz C，Terzioglu E，Topalak O，et al.Serum transforming growth factor-beta1（TGF-beta1）in patients with cirrhosis，chronic hepatitis B and chronic hepatitis C［corrected］［J］. Eur Cytokine Netw，2004，15（2）:112-116.

［19］Arany PR，Flanders KC，DeGraff W，et al.Absence of Smad3 confers radioprotection through modulation of ERK-MAPK in primary dermal fibroblasts［J］.J Dermatol Sci，2007，48（1）: 35-42.

［20］Ruiz-Ortega M，Rodríguez-Vita J，Sanchez-Lopez E，et al. TGF-beta signaling in vascular fibrosis［J］.Cardiovasc Res，2007，74（2）:196-206.

［21］Lauer-FieldsJL，JuskaD，FieldsGB.Fields，Matrix metalloproteinases and collagen catabolism［J］.Biopolymers，2002，66（1）:19-32.

［22］Desmoulière A，Geinoz A，Gabbiani F，et al.Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts［J］.J Cell Biol，1993，122（1）:103-11.

［23］Yokose U，Hachiya A，Sriwiriyanont P，et al.The endogenous protease inhibitor TIMP-1 mediates protection and recovery from cutaneous photodamage［J］.J Invest Dermatol，2012，132（12）:2800-2809.

Received date：2014-12-17 Revised date：2015-02-05

Editor：ZHANG Hui-juan

*Corresponding author:LIU Su，Department of Plastic and Aesthetic Surgery，The Affiliated Hospital of Medical School，Qingdao University，No.111 Yan Er Dao Road，Qingdao 266071，P.R.China.E-mail:liusuqingdao@163.com